The present invention relates to increasing bacterial toxin production using methods and compositions that reduce, or eliminate, the accumulation of intracellular and extracellular toxin expression inhibitors. Specifically, the present invention related to methods and compositions for reducing or elimination the accumulation of Bordetella species toxin expression inhibitors. More specifically, the present invention relates to the high yield production of pertussis toxin, pertactin, adenylate cyclase toxin-hemolysin, filamentous hemagglutinin and other toxins.
Pertussis toxin (PT) is one of the various components produced by virulent B. pertussis, the microorganism that causes whooping cough. Whooping cough is a serious infection of the respiratory system that at one time was responsible for the death of 5,000 to 10,000 people in the United States each year. Since the advent of the whooping cough vaccine the number of whooping cough related deaths has been reduced to less than 20 annually. Currently, about 50% of all whooping cough infections occur in children less than 1 year old, and only 15% occur in children over 15 years old.
PT is a major protective antigen in the vaccine against whooping cough. Other components of interest produced by B. pertussis are filamentous hemagglutinin, heat labile toxin, adenylate cyclase and the like, which may also play important role as protective antigens. Large-scale production of these components, which are useful as diagnostic or chemical reagents and in the preparation of vaccines, requires large-scale cultivation of the microorganism. However, B. pertussis is a fastidious organism that has proved difficult to grow in large fermentors. Older methods for the culture of B. pertussis employ cultivation in stationary culture or in fermentors. Growth in a stationary culture is labor intensive, while cultivation on a fermentation scale requires vortex stirring and surface aeration. As a result, the effective volume of the fermentor is reduced and modification of the fermentor for growth of pertussis is often necessary. Furthermore, the quantities of PT produced during fermentation under these conditions are variable and often low.
U.S. Pat. No. 5,338,670 discloses a method for the production of B. pertussis in the presence of an iron salt, namely ferrous sulfate. While high iron content supports greater bacterial growth, it suppresses the production of PT. By adjusting the iron content of modified Stainer-Scholte media to 10% of the recommended concentration, the production of PT was optimized.
The present invention seeks to improve the yield of PT obtained from B. pertussis by (1) introducing a soluble salt into the growth medium that sequesters sulfate (SO42−) and/or (2) employing a B. pertussis cysteine desulfinase knockout mutant.